JP7432886B2 - New aortic aneurysm marker - Google Patents
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Description
本発明は、大動脈瘤の診断を補助する臨床検査薬の技術分野に関する。詳細には、大動脈瘤のバイオマーカー、それを用いる大動脈瘤の検出剤、大動脈瘤検出キット、及び大動脈瘤の検出方法に関するものである。 The present invention relates to the technical field of clinical test drugs that assist in the diagnosis of aortic aneurysms. Specifically, the present invention relates to an aortic aneurysm biomarker, an aortic aneurysm detection agent using the same, an aortic aneurysm detection kit, and an aortic aneurysm detection method.
大動脈瘤は、大動脈の一部の壁が全周性又は局所性に(径)拡大又は突出した状態であり、瘤の発生部位により胸部大動脈では胸部大動脈瘤、胸部と腹部に連続する胸腹部大動脈瘤、腹部では腹部大動脈瘤と称される(非特許文献１)。大動脈瘤発症のピーク年齢は70～80代であり、人口の高齢化により近年増加しているが、検査しない限り大動脈瘤が発見されることはなく、ほぼ無症状で進行し、破裂に至ると生命の危機につながる重篤な病態となる。しかし、適切な治療を行うことにより破裂を回避することが可能であるため、大動脈瘤を早期に検出することは非常に重要である。 An aortic aneurysm is a condition in which part of the wall of the aorta expands (diameter) or protrudes circumferentially or locally. In the abdomen, it is called an abdominal aortic aneurysm (Non-Patent Document 1). The peak age at which an aortic aneurysm develops is between the ages of 70 and 80, and the number has increased in recent years due to the aging of the population. However, an aortic aneurysm is not discovered unless it is examined and progresses almost asymptomatically, leading to rupture. It becomes a serious condition that can lead to a life-threatening condition. However, early detection of aortic aneurysms is very important, as rupture can be avoided with appropriate treatment.
胸部大動脈瘤は、健康診断などで胸部レントゲン写真を撮った時に偶然発見されることがほとんどであり、腹部大動脈瘤の場合は、胃潰瘍や胆石症などの消化器疾患を診断するために腹部を触診した際に、拍動するしこりとして発見されたり、消化管や腎臓、前立腺などの腹部エコー(超音波)の検査中あるいは胸部腹部のMRI検査中に偶然に発見されたりすることが多い。 Thoracic aortic aneurysms are most often discovered incidentally when a chest X-ray is taken during a medical checkup, while abdominal aortic aneurysms are discovered by palpation of the abdomen to diagnose gastrointestinal diseases such as gastric ulcers and cholelithiasis. It is often discovered as a pulsating lump, or discovered incidentally during an abdominal ultrasound (ultrasound) examination of the gastrointestinal tract, kidneys, prostate, etc., or during an MRI examination of the chest and abdomen.
一方、大動脈瘤のマーカーとしては、Dダイマー、CRP、WBC、血漿ホモシステイン等が知られている(非特許文献２及び３)。また、Peroxiredoxin-1(以下、「PRDX1」と称する)が心血管疾患や大動脈瘤のバイオマーカーとして知られている(非特許文献４及び５)。 On the other hand, D-dimer, CRP, WBC, plasma homocysteine, etc. are known as markers for aortic aneurysm (Non-patent Documents 2 and 3). Additionally, Peroxiredoxin-1 (hereinafter referred to as "PRDX1") is known as a biomarker for cardiovascular disease and aortic aneurysm (Non-Patent Documents 4 and 5).
しかし、いずれも、感度、特異度や疾患特異性の面から十分なものとは言えない。そのため、大動脈瘤を特異的且つ高感度に検出可能なバイオマーカーの開発が望まれている。 However, none of these methods can be said to be sufficient in terms of sensitivity, specificity, and disease specificity. Therefore, it is desired to develop a biomarker that can specifically and sensitively detect aortic aneurysms.
本発明は、上記の現状に鑑みてなされたものであり、大動脈瘤を高感度に検出できる新規なバイオマーカーを提供することを目的としている。 The present invention has been made in view of the above-mentioned current situation, and aims to provide a novel biomarker that can detect aortic aneurysms with high sensitivity.
本発明者らは、感度や特異性に優れた大動脈瘤マーカーの提供を目的として鋭意検討し、胸部大動脈瘤組織を用いたプロテオーム解析を実施した。健常者大動脈組織と胸部大動脈瘤患者組織のプロテオームデータの比較から得られた疾患組織で顕著に変動する因子から、血液中において大動脈瘤を高感度、特異的に検出可能な新たなマーカーを見出した。 The present inventors conducted intensive studies with the aim of providing an aortic aneurysm marker with excellent sensitivity and specificity, and conducted proteome analysis using thoracic aortic aneurysm tissue. A new marker that can detect aortic aneurysms with high sensitivity and specificity in the blood has been discovered from factors that vary significantly in diseased tissue obtained by comparing proteome data of healthy aortic tissue and tissue of thoracic aortic aneurysm patients. .
また、健常者及び胸部大動脈瘤患者の血清を用いたプロテオーム解析を実施した。得られた血清のプロテオミクスデータを組織プロテオミクスのデータと照合し、組織及び血清の両方において大動脈瘤で顕著な増加を示すタンパク質を、大動脈瘤を高感度、特異的に検出可能な新たなマーカーとして見出し、本発明を完成するに至った。 In addition, proteome analysis was performed using serum from healthy subjects and patients with thoracic aortic aneurysm. By comparing the obtained serum proteomics data with tissue proteomics data, we discovered a protein that shows a significant increase in aortic aneurysms in both tissue and serum as a new marker that can detect aortic aneurysms with high sensitivity and specificity. , we have completed the present invention.
すなわち、本発明は以下のような構成から成るものである。
（１）対象から得られた試料において、以下の1)～3)のいずれか1つ以上のタンパク質を測定する工程を含む、大動脈瘤の検出方法。
1) MXRA5(Matrix-remodeling-associated protein 5)タンパク質、
2) TALDO1(Transaldolase)タンパク質
3) METRNL(Meteorin-like protein)タンパク質。
（２）前記対象が、大動脈瘤の予防又は治療を必要とする動物であり、前記タンパク質を指標として、大動脈瘤の予防薬又は治療薬の効果を評価又は判定する、（１）記載の方法。
（３）対象から得られた試料において、以下の1)～3)のいずれか1つ以上のタンパク質を測定する工程を含む、大動脈瘤を将来発症するリスクを有する対象を検出又はスクリーニングするための方法。
1) MXRA5(Matrix-remodeling-associated protein 5)タンパク質、
2) TALDO1(Transaldolase)タンパク質
3) METRNL(Meteorin-like protein)タンパク質。
（４）前記大動脈瘤が胸部大動脈瘤、胸腹部大動脈瘤及び/又は腹部大動脈瘤である、（１）～（３）のいずれか１記載の方法。
（５）試料が、血清、血漿、血液、尿及び唾液から成る群より選択される、（１）～（４）のいずれか１記載の方法。
（６）前記対象がヒトである、（１）～（５）のいずれか１記載の方法。
（７）測定方法が免疫測定法又は質量分析法である、（１）～（６）のいずれか１記載の方法。
（８）以下の1)～3)のいずれか1つ以上のタンパク質を含む大動脈瘤の検出マーカー。
That is, the present invention consists of the following configuration.
(1) A method for detecting an aortic aneurysm, comprising the step of measuring one or more of the following proteins in 1) to 3) in a sample obtained from a subject.
1) MXRA5 (Matrix-remodeling-associated protein 5) protein,
2) TALDO1 (Transaldolase) protein
3) METRNL (Meteorin-like protein) protein.
(2) The method according to (1), wherein the subject is an animal in need of prevention or treatment of aortic aneurysm, and the effect of a prophylactic or therapeutic agent for aortic aneurysm is evaluated or determined using the protein as an index.
(3) A method for detecting or screening a subject at risk of developing an aortic aneurysm in the future, including the step of measuring one or more of the following proteins in 1) to 3) in a sample obtained from the subject. Method.
1) MXRA5 (Matrix-remodeling-associated protein 5) protein,
2) TALDO1 (Transaldolase) protein
3) METRNL (Meteorin-like protein) protein.
(4) The method according to any one of (1) to (3), wherein the aortic aneurysm is a thoracic aortic aneurysm, a thoracoabdominal aortic aneurysm, and/or an abdominal aortic aneurysm.
(5) The method according to any one of (1) to (4), wherein the sample is selected from the group consisting of serum, plasma, blood, urine, and saliva.
(6) The method according to any one of (1) to (5), wherein the subject is a human.
(7) The method according to any one of (1) to (6), wherein the measurement method is immunoassay or mass spectrometry.
(8) An aortic aneurysm detection marker containing one or more of the following proteins 1) to 3).
1) MXRA5(Matrix-remodeling-associated protein 5)タンパク質、
2) TALDO1(Transaldolase)タンパク質
3) METRNL(Meteorin-like protein)タンパク質。
（９）前記大動脈瘤が胸部大動脈瘤、胸腹部大動脈瘤及び/又は腹部大動脈瘤である、（８）記載の検出マーカー。
（１０）（８）又は（９）記載の検出マーカーを測定するための抗体又はアプタマーを含む大動脈瘤の検出剤。
（１１）前記抗体がモノクローナル抗体及び/又はポリクローナル抗体である、（１０）記載の検出剤。
（１２）（１０）又は（１１）記載の検出剤を含む、大動脈瘤の検出キット。
（１３）大動脈瘤の検出に使用される試料が、血清、血漿、血液、尿及び唾液から成る群より選択される、（８）～（１２）のいずれか１記載の検出マーカー、検出剤又は検出キット。
（１４）大動脈瘤の予防薬又は治療薬のスクリーニングのための、（８）又は（９）記載の検出マーカーの使用。
（１５）大動脈瘤を将来発症するリスクを有する対象をin vitroで検出又はスクリーニングするための、（８）又は（９）記載の検出マーカーの使用。
1) MXRA5 (Matrix-remodeling-associated protein 5) protein,
2) TALDO1 (Transaldolase) protein
3) METRNL (Meteorin-like protein) protein.
(9) The detection marker according to (8), wherein the aortic aneurysm is a thoracic aortic aneurysm, a thoracoabdominal aortic aneurysm, and/or an abdominal aortic aneurysm.
(10) An aortic aneurysm detection agent comprising an antibody or aptamer for measuring the detection marker described in (8) or (9).
(11) The detection agent according to (10), wherein the antibody is a monoclonal antibody and/or a polyclonal antibody.
(12) An aortic aneurysm detection kit comprising the detection agent described in (10) or (11).
(13) The detection marker, detection agent, or detection kit.
(14) Use of the detection marker described in (8) or (9) for screening for preventive or therapeutic agents for aortic aneurysm.
(15) Use of the detection marker described in (8) or (9) for in vitro detection or screening of subjects who have a risk of developing an aortic aneurysm in the future.
本発明により見出したMXRA5、TALDO1及びMETRNLのうちいずれか1つ以上のタンパク質を大動脈瘤のマーカーとして用いることにより、大動脈瘤の発症を高感度で特異的に検出することができる。 By using any one or more proteins among MXRA5, TALDO1, and METRNL discovered by the present invention as a marker for aortic aneurysm, the onset of aortic aneurysm can be detected specifically and with high sensitivity.
また、本発明による大動脈瘤の検出剤及び検出キットは胸部大動脈瘤、胸腹部大動脈瘤、腹部大動脈瘤ともに、その発症の早期診断の補助に使用することが可能であり、当該疾患の早期検出により疾患の重症化の回避に貢献可能である。さらに大動脈瘤の予防薬や治療薬の開発、評価にも使用することが可能と考えられ、大動脈瘤の予防や治療にも利用可能である。 Furthermore, the aortic aneurysm detection agent and detection kit according to the present invention can be used to assist in the early diagnosis of the onset of thoracic aortic aneurysm, thoracoabdominal aortic aneurysm, and abdominal aortic aneurysm. It can contribute to avoiding the severity of the disease. Furthermore, it is thought that it can be used for the development and evaluation of preventive and therapeutic drugs for aortic aneurysms, and can also be used for the prevention and treatment of aortic aneurysms.
本発明者等は、大動脈瘤マーカーの同定を目的として、健常者大動脈組織14例、胸部大動脈瘤患者組織29例から採取した大動脈中膜平滑筋層組織を用いてプロテオーム解析を以下のように行った。 For the purpose of identifying aortic aneurysm markers, the present inventors performed proteome analysis as follows using aortic media smooth muscle tissue collected from 14 healthy subjects and 29 thoracic aortic aortic aneurysm patient tissues. Ta.
・組織の破砕とトリプシン消化
採取後、凍結した組織はビーズ破砕機を用いて破砕し、Lys-C(Lysyl Endopeptidase)、Trypsinを添加して37℃で一晩インキュベーションにより消化した。得られた消化ペプチド（以下、トリプシン消化ペプチド）はC18(オクタデシルシリル)カラムを用いて脱塩し、解析サンプルとした。
- Tissue crushing and trypsin digestion After collection, the frozen tissue was crushed using a bead crusher, Lys-C (Lysyl Endopeptidase) and Trypsin were added, and it was digested by incubation at 37°C overnight. The obtained digested peptide (hereinafter referred to as tryptic digested peptide) was desalted using a C18 (octadecylsilyl) column and used as an analysis sample.
・解析
解析サンプルはナノLC(日立Nano Frontier、日立ハイテクノロジーズ社)で分離した後、連結されたTripleTOF 5600(SCIEX社)でMS/MS解析を行った。測定データからMascot database search(Matrix Science社)でタンパク質を同定し、LC-MSデータを集積して用いる比較定量プロテオーム解析ソフトである2DICAL(2-Dimensional Image Converted Analysis of Liquid chromatography-mass spectrometry、三井情報株式会社)を用いて定量を行い、非血管疾患群の大動脈と胸部大動脈瘤群の大動脈瘤の中膜平滑筋組織間で差のあるタンパク質の探索を行った。その結果、正常大動脈群に対し胸部大動脈瘤群で顕著な増加を示すタンパク質としてMeteorin-like protein(以下、「METRNL」と称する)を同定した。
-Analysis After the analysis sample was separated using Nano LC (Hitachi Nano Frontier, Hitachi High-Technologies), it was subjected to MS/MS analysis using a connected TripleTOF 5600 (SCIEX). 2DICAL (2-Dimensional Image Converted Analysis of Liquid chromatography-mass spectrometry, Mitsui Information Co., Ltd.) is a comparative quantitative proteome analysis software that uses Mascot database search (Matrix Science) to identify proteins from measurement data and accumulates LC-MS data. Co., Ltd.) to search for proteins that differ between the media smooth muscle tissue of the aorta in the non-vascular disease group and the aortic aneurysm in the thoracic aortic aneurysm group. As a result, we identified Meteorin-like protein (hereinafter referred to as "METRNL") as a protein that showed a marked increase in the thoracic aortic aneurysm group compared to the normal aorta group.
・血清の分画と解析
健常者及び胸部大動脈瘤患者の血清を用いたプロテオーム解析を以下のように行った。
血清は複数例を混合した後、超遠心機を用いて比重によりLDL(Low-density lipoprotein)/VLDL(Very low-density lipoprotein)分画、HDL(High-density lipoprotein)分画、Protein分画の3分画に分けた。Protein分画は採取後、血中多量タンパク質除去Sepproアフィニティカラム(Sigma-Aldrich社)にて多量タンパク質を除去した。その後、各分画をLys-C(Lysyl Endopeptidase)、Trypsinを添加し、37℃で一晩インキュベーションして消化した。得られたトリプシン消化ペプチドはC18(オクタデシルシリル)カラムを用いて脱塩し、解析サンプルとした。
- Fractionation and analysis of serum Proteome analysis using serum from healthy subjects and patients with thoracic aortic aneurysm was performed as follows.
After mixing multiple samples of serum, the serum is divided into LDL (Low-density lipoprotein)/VLDL (Very low-density lipoprotein) fraction, HDL (High-density lipoprotein) fraction, and Protein fraction according to specific gravity using an ultracentrifuge. Divided into 3 fractions. After the protein fraction was collected, large amounts of proteins in blood were removed using a Seppro affinity column (Sigma-Aldrich). Thereafter, each fraction was digested by adding Lys-C (Lysyl Endopeptidase) and Trypsin and incubating at 37°C overnight. The obtained trypsin-digested peptides were desalted using a C18 (octadecylsilyl) column and used as analysis samples.
解析サンプルはナノLC(Eksigent EkspertTMnanoLC 425、SCIEX社)で分離した後、連結されたTripleTOF 5600(SCIEX社)でMS/MS解析を行った。測定データからMascot database search (Matrix science社)でペプチド及びタンパク質情報を取得し、Skylineソフトウェア(University of Washington MacCoss Lab)を用いてMRMメソッド(MRM測定を行うためのプログラム)を作成した。その後、健常者血清及び胸部大動脈瘤患者血清から調製した解析サンプルについてMRM法による測定を行い、得られたデータをMultiQuantソフトウェア(SCIEX社)で解析することで、各トリプシン消化ペプチドのピーク面積を比較定量した。 The analysis sample was separated using a nanoLC (Eksigent Ekspert TM nanoLC 425, SCIEX), and then subjected to MS/MS analysis using a connected TripleTOF 5600 (SCIEX). Peptide and protein information was obtained from the measurement data using Mascot database search (Matrix science), and an MRM method (program for performing MRM measurement) was created using Skyline software (University of Washington MacCoss Lab). After that, analysis samples prepared from serum from healthy subjects and serum from patients with thoracic aortic aneurysm were measured using the MRM method, and the obtained data was analyzed using MultiQuant software (SCIEX) to compare the peak areas of each tryptic peptide. Quantitated.
得られた血清のプロテオミクスデータを組織プロテオミクスのデータと照合し、組織及び血清の両方において胸部大動脈瘤患者で顕著な増加を示すタンパク質としてMatrix-remodeling-associated protein 5(以下、「MXRA5」と称する)、Transaldolase(以下、「TALDO1」と称する)を同定し、本発明を完成するに至った。 The obtained serum proteomics data was compared with tissue proteomics data, and Matrix-remodeling-associated protein 5 (hereinafter referred to as "MXRA5") was identified as a protein that shows a significant increase in thoracic aortic aneurysm patients in both tissue and serum. , Transaldolase (hereinafter referred to as "TALDO1") was identified, and the present invention was completed.
以上の通り、MXRA5、TALDO1及びMETRNLはいずれも、胸部大動脈瘤患者由来の大動脈中膜組織又は血清若しくは血漿において、健常者と比較して顕著に発現が亢進していることが確認されている。MXRA5、TALDO1、METRNLをそれぞれ単独でマーカーとして使用することにより、大動脈瘤の検出が可能である。また、これらのうち2つ又は3つ全てを組み合わせてマーカーとして使用してもよい。 As described above, it has been confirmed that the expression of MXRA5, TALDO1, and METRNL is significantly increased in aortic media tissue, serum, or plasma derived from thoracic aortic aneurysm patients compared to healthy subjects. Aortic aneurysms can be detected by using MXRA5, TALDO1, and METRNL individually as markers. Further, two or all three of these may be used in combination as a marker.
以上に説明するように、本発明は、MXRA5、TALDO1及びMETRNLのうちいずれか1つ以上を大動脈瘤の検出マーカーとすることを特徴とする。 As explained above, the present invention is characterized by using any one or more of MXRA5, TALDO1, and METRNL as a detection marker for aortic aneurysm.
本発明において、「マーカー」又は「バイオマーカー」は、対象から得られた試料の測定用の標的として使用される分子のことをいう。 In the present invention, "marker" or "biomarker" refers to a molecule used as a target for measurement of a sample obtained from a subject.
本発明に係る大動脈瘤の検出マーカーは、MXRA5、TALDO1及びMETRNLのうちいずれか1つ以上を含む。 The aortic aneurysm detection marker according to the present invention includes any one or more of MXRA5, TALDO1, and METRNL.
ヒトMXRA5は、UniProtのアクセション番号がQ9NR99で表される分泌性のタンパク質である(https://www.uniprot.org/uniprot/Q9NR99)。ヒトMXRA5のアミノ酸配列を配列番号１に示す。配列番号１に示すアミノ酸配列において、第1番目～第26番目のアミノ酸配列はシグナルペプチドであり、第27番目～第2828番目のアミノ酸配列は成熟型タンパク質である。MXRAファミリーは、MXRA5、MXRA7、MXRA8の3つの遺伝子から構成され、細胞接着やマトリックスリモデリングに関わると考えられている機能未知のタンパク質だが、抗炎症、抗線維化分子として機能することが報告されている。遺伝子からはMXRA5の分子量は312kDaと予想される。MXRA5は、幾つかの哺乳類には見つかっているが、マウスやラットには見つかっていない(Jonay Poveda, Ana B. Sanz, Beatriz Fernandez-Fernandez, Susana Carrasco, Marta Ruiz-Ortega, Pablo Cannata-Ortiz, Alberto Ortiz, Maria D. Sanchez-Nino. MXRA5 is a TGF-β1-regulated human protein with anti-inflammatory and anti-fibrotic properties. J. Cell. Mol. Med. 2017; Vol 21, No 1, 154-164)。 Human MXRA5 is a secreted protein whose UniProt accession number is Q9NR99 (https://www.uniprot.org/uniprot/Q9NR99). The amino acid sequence of human MXRA5 is shown in SEQ ID NO: 1. In the amino acid sequence shown in SEQ ID NO: 1, the 1st to 26th amino acid sequences are the signal peptide, and the 27th to 2828th amino acid sequences are the mature protein. The MXRA family consists of three genes, MXRA5, MXRA7, and MXRA8, and is a protein whose function is unknown and is believed to be involved in cell adhesion and matrix remodeling, but it has been reported that it functions as an anti-inflammatory and anti-fibrotic molecule. ing. Based on the gene, the molecular weight of MXRA5 is predicted to be 312kDa. MXRA5 has been found in several mammals, but not mice or rats (Jonay Poveda, Ana B. Sanz, Beatriz Fernandez-Fernandez, Susana Carrasco, Marta Ruiz-Ortega, Pablo Cannata-Ortiz, Alberto Ortiz, Maria D. Sanchez-Nino. MXRA5 is a TGF-β1-regulated human protein with anti-inflammatory and anti-fibrotic properties. J. Cell. Mol. Med. 2017; Vol 21, No 1, 154-164).
本発明において、MXRA5タンパク質には、配列番号１に示すアミノ酸配列における第27番目～第2828番目のアミノ酸配列に対して70％以上、75％以上、80％以上、85％以上、90％以上、より好ましくは95％以上、96％以上、97％以上、98％以上、99％以上、最も好ましくは100％の配列同一性を有するアミノ酸配列から成り、且つ細胞接着機能、マトリックスリモデリング機能、抗炎症機能及び抗線維化機能のうちいずれか1つ以上の機能を有するか、あるいは機能欠損変異体等の当該機能を有しないタンパク質が含まれ、さらにそのようなタンパク質の多量体(二量体以上)も含まれる。 In the present invention, the MXRA5 protein includes 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, More preferably, it consists of an amino acid sequence having sequence identity of 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, and most preferably 100%, and has cell adhesion function, matrix remodeling function, and anti-inflammatory function. It includes proteins that have one or more of inflammatory and antifibrotic functions, or that do not have such functions, such as function-defective mutants, and also include multimers (dimers or more) of such proteins. ) is also included.
ヒトTALDO1は、UniProtのアクセション番号がP37837で表される分泌性のタンパク質である(https://www.uniprot.org/uniprot/P37837)。ヒトTALDO1のアミノ酸配列を配列番号２に示す。TALDO1は、ペントースリン酸経路における代謝物の均衡に重要な酵素の1つである。 Human TALDO1 is a secreted protein whose UniProt accession number is P37837 (https://www.uniprot.org/uniprot/P37837). The amino acid sequence of human TALDO1 is shown in SEQ ID NO:2. TALDO1 is one of the enzymes important for metabolite balance in the pentose phosphate pathway.
本発明において、TALDO1タンパク質には、配列番号２に示すアミノ酸配列に対して70％以上、75％以上、80％以上、85％以上、90％以上、より好ましくは95％以上、96％以上、97％以上、98％以上、99％以上、最も好ましくは100％の配列同一性を有するアミノ酸配列から成り、且つペントースリン酸経路における代謝物均衡機能を有するか、あるいは機能欠損変異体等の当該機能を有しないタンパク質が含まれ、さらにそのようなタンパク質の多量体(二量体以上)も含まれる。 In the present invention, the TALDO1 protein includes 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, more preferably 95% or more, 96% or more, with respect to the amino acid sequence shown in SEQ ID NO: 2. Consists of an amino acid sequence with sequence identity of 97% or more, 98% or more, 99% or more, most preferably 100%, and has a metabolite balance function in the pentose phosphate pathway, or a mutant lacking the function, etc. It includes proteins that do not have a protein, and also includes multimers (dimers or more) of such proteins.
ヒトMETRNLは、UniProtのアクセション番号がQ641Q3で表される分泌性のタンパク質である(https://www.uniprot.org/uniprot/Q641Q3）。ヒトMETRNLのアミノ酸配列を配列番号３に示す。配列番号３に示すアミノ酸配列において、第1番目～第45番目のアミノ酸配列はシグナルペプチドであり、第46番目～第311番目のアミノ酸配列は成熟型タンパク質である。METRNLは、新しいアディポサイトカインの1つであり、約40％のアミノ酸が一致するMeteorinのホモログである。ホモログのMeteorinとは異なり、METRNLはマウスの様々な組織で異なるレベルで発現しており、METRNLは神経栄養性因子として働くことや、白色脂肪細胞の褐色化に関わることが報告されている(Nishino J, Yamashita K, Hashiguchi H, Fujii H, Shimazaki T, Hamada H. Meteorin: a secreted protein that regulates glial cell differentiation and promotes axonal extension. EMBO J 2004; 23: 1998-2008.及びSi-li ZHENG1, Zhi-yong LI, Jie SONG, Jian-min LIU, Chao-yu MIAO. Metrnl: a secreted protein with new emerging functions. Acta Pharmacologica Sinica. 2016; 37: 571-579)。 Human METRNL is a secreted protein whose UniProt accession number is Q641Q3 (https://www.uniprot.org/uniprot/Q641Q3). The amino acid sequence of human METRNL is shown in SEQ ID NO:3. In the amino acid sequence shown in SEQ ID NO: 3, the 1st to 45th amino acid sequence is a signal peptide, and the 46th to 311th amino acid sequence is a mature protein. METRNL is one of the new adipocytokines and is a homologue of Meteorin with approximately 40% amino acid identity. Unlike its homologue Meteorin, METRNL is expressed at different levels in various tissues of mice, and it has been reported that METRNL acts as a neurotrophic factor and is involved in the browning of white adipocytes (Nishino J, Yamashita K, Hashiguchi H, Fujii H, Shimazaki T, Hamada H. Meteorin: a secreted protein that regulates glial cell differentiation and promotes axonal extension. EMBO J 2004; 23: 1998-2008. and Si-li ZHENG1, Zhi- yong LI, Jie SONG, Jian-min LIU, Chao-yu MIAO. Metrnl: a secreted protein with new emerging functions. Acta Pharmacologica Sinica. 2016; 37: 571-579).
本発明において、METRNLタンパク質には、配列番号３に示すアミノ酸配列における第46番目～第311番目のアミノ酸配列に対して70％以上、75％以上、80％以上、85％以上、90％以上、より好ましくは95％以上、96％以上、97％以上、98％以上、99％以上、最も好ましくは100％の配列同一性を有するアミノ酸配列から成り、且つ神経栄養性因子機能及び白色脂肪細胞の褐色化機能のうちいずれか1つ以上の機能を有するか、あるいは機能欠損変異体等の当該機能を有しないタンパク質が含まれ、さらにそのようなタンパク質の多量体(二量体以上)も含まれる。 In the present invention, the METRNL protein includes 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, More preferably, it consists of an amino acid sequence having sequence identity of 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, and most preferably 100%, and is highly effective for neurotrophic factor function and white adipocyte function. Includes proteins that have one or more of the browning functions or do not have such functions, such as function-deficient mutants, and also include multimers (dimers or more) of such proteins. .
なお、本発明に係るマーカーをC反応性蛋白(CRP)、Dダイマー、白血球数(WBC)、血漿ホモシステイン、マトリックスメタロプロテアーゼ-9(MMP-9)等の既知の大動脈瘤マーカーと組み合わせて使用することも可能である。 Note that the marker according to the present invention is used in combination with known aortic aneurysm markers such as C-reactive protein (CRP), D-dimer, white blood cell count (WBC), plasma homocysteine, and matrix metalloproteinase-9 (MMP-9). It is also possible to do so.
本発明に係るマーカーにより検出可能な大動脈瘤の発症部位は特に限定されない。大動脈基部、上行大動脈、大動脈弓部、下行大動脈のいずれかに発症した胸部大動脈瘤、胸腹部大動脈瘤、腹部大動脈瘤のいずれも検出可能である。 The site of onset of an aortic aneurysm that can be detected by the marker according to the present invention is not particularly limited. It is possible to detect thoracic aortic aneurysms, thoracoabdominal aortic aneurysms, and abdominal aortic aneurysms that develop in any of the aortic root, ascending aorta, aortic arch, or descending aorta.
本発明において、大動脈瘤を検出する方法は、MXRA5、TALDO1及びMETRNLのうちいずれか1つ以上のタンパク質を測定する工程を含む。試料中のMXRA5、TALDO1及びMETRNLのうちいずれか1つ以上のタンパク質を測定するに際しては、糖タンパク質としてのMXRA5、TALDO1及びMETRNLのうちいずれか1つ以上のタンパク質を測定しても良く、また、前処理により糖鎖、リン酸等の翻訳後修飾を除去した後にMXRA5、TALDO1及びMETRNLのうちいずれか1つ以上のタンパク質のアミノ酸配列部分を測定しても良い。 In the present invention, the method for detecting an aortic aneurysm includes the step of measuring one or more proteins among MXRA5, TALDO1, and METRNL. When measuring one or more proteins among MXRA5, TALDO1, and METRNL in a sample, one or more proteins among MXRA5, TALDO1, and METRNL as glycoproteins may be measured, and After post-translational modifications such as sugar chains and phosphates are removed by pretreatment, the amino acid sequence portion of any one or more proteins among MXRA5, TALDO1, and METRNL may be measured.
大動脈瘤を検出する方法は、対象から得られた試料において本発明に係るマーカーを測定することで、当該対象の大動脈瘤を検出することができる。従って、大動脈瘤を検出する方法は、大動脈瘤の診断、検査、評価又は判定方法、あるいは大動脈瘤を検出するためのin vitroにおけるデータ収集方法等ということができる。 A method for detecting an aortic aneurysm can detect an aortic aneurysm in a subject by measuring the marker according to the present invention in a sample obtained from the subject. Therefore, a method for detecting an aortic aneurysm can be referred to as a method for diagnosing, testing, evaluating, or determining an aortic aneurysm, or an in vitro data collection method for detecting an aortic aneurysm.
大動脈瘤を検出する方法の被験対象となり得る動物としては、例えばヒト、サル、ウシ、ブタ、ウマ、イヌ、ネコ、ヒツジ、ヤギ、ウサギ、ハムスター、モルモット、マウス、ラット等の哺乳類が挙げられるが、好ましくはヒトである。 Examples of animals that can be used as test subjects for the method for detecting aortic aneurysms include mammals such as humans, monkeys, cows, pigs, horses, dogs, cats, sheep, goats, rabbits, hamsters, guinea pigs, mice, and rats. , preferably human.
また、大動脈瘤を検出する方法において使用する試料としては、例えば、血清、血漿、血液、尿及び唾液等が挙げられる。 Further, examples of samples used in the method for detecting an aortic aneurysm include serum, plasma, blood, urine, and saliva.
本発明においては、例えば、大動脈瘤が疑われる患者から採取された試料において、in vitroでMXRA5、TALDO1及びMETRNLのうちいずれか1つ以上のタンパク質レベルを測定することにより、大動脈瘤の発症可能性を判断することができる。患者検体中のMXRA5、TALDO1及びMETRNLのうちいずれか1つ以上のタンパク質レベルを健常者のレベルと比較し、患者検体中のMXRA5、TALDO1及びMETRNLのうちいずれか1つ以上のタンパク質レベルが健常者のレベルに比べて高い場合、大動脈のいずれかの部位で将来、大動脈瘤を発症する可能性あるいは現在大動脈瘤が進行している可能性が高いと判断できる。 In the present invention, for example, by measuring the protein level of any one or more of MXRA5, TALDO1, and METRNL in a sample collected from a patient suspected of having an aortic aneurysm, the possibility of developing an aortic aneurysm can be determined. can be judged. The protein level of any one or more of MXRA5, TALDO1, and METRNL in the patient sample is compared with the level of healthy subjects, and the protein level of any one or more of MXRA5, TALDO1, and METRNL in the patient sample is compared to that of healthy subjects. If it is higher than the level of , it can be determined that there is a high possibility that an aortic aneurysm will develop in the future in some part of the aorta, or that there is a high possibility that the aortic aneurysm is currently progressing.
具体的には、大動脈瘤を検出する方法に準じて、本発明は、対象から得られた試料において、本発明に係るマーカーを測定することで、大動脈瘤を将来発症するリスクを有する対象を検出又はスクリーニングする方法をさらに含む。 Specifically, in accordance with a method for detecting an aortic aneurysm, the present invention detects a subject at risk of developing an aortic aneurysm in the future by measuring the marker according to the present invention in a sample obtained from the subject. or a method of screening.
また、大動脈瘤を発症する可能性あるいは現在大動脈瘤が進行している可能性が高いと判断するための各マーカーのカットオフ値としては、限定されるものではないが、例えば、以下のマーカーのカットオフ値は、実施例に示すデータを基に作成したROC曲線から得られたものである。 In addition, cut-off values for each marker for determining that there is a high possibility of developing an aortic aneurysm or that the aortic aneurysm is currently progressing are not limited, but include, but are not limited to, the following markers: The cutoff value was obtained from the ROC curve created based on the data shown in the Examples.
(1) MXRA5
・胸部大動脈瘤又は腹部大動脈瘤：
血漿中のカットオフ値：26.1 ng/mL（感度55.9％、特異度80.0％）
(2) METRNL
・胸部大動脈瘤：
血清中のカットオフ値：1466.4 pg/mL（感度64.3％、特異度88.8％）
・腹部大動脈瘤：
血清中のカットオフ値：1477.5 pg/ml（感度47.4％、特異度92.3％）
(1) MXRA5
・Thoracic aortic aneurysm or abdominal aortic aneurysm:
Cutoff value in plasma: 26.1 ng/mL (sensitivity 55.9%, specificity 80.0%)
(2) METRNL
・Thoracic aortic aneurysm:
Serum cutoff value: 1466.4 pg/mL (sensitivity 64.3%, specificity 88.8%)
・Abdominal aortic aneurysm:
Serum cutoff value: 1477.5 pg/ml (sensitivity 47.4%, specificity 92.3%)
このようにカットオフ値に基づいて、患者検体中のMXRA5、TALDO1及びMETRNLのうちいずれか1つ以上のタンパク質レベルがカットオフ値に比べて高い場合、胸部大動脈瘤又は腹部大動脈瘤を発症する可能性あるいは現在胸部大動脈瘤又は腹部大動脈瘤が進行している可能性が高いと判断できる。 Based on the cutoff value, if the protein level of any one or more of MXRA5, TALDO1, and METRNL in the patient sample is higher than the cutoff value, there is a possibility that thoracic aortic aneurysm or abdominal aortic aneurysm will develop. It can be determined that there is a high possibility that a thoracic aortic aneurysm or abdominal aortic aneurysm is currently progressing.
本発明に係るマーカーは、大動脈瘤の予防薬又は治療薬のスクリーニングや、大動脈瘤の予防薬又は治療薬の効果の評価や判定のために使用することも可能である。例えば、大動脈瘤の予防薬又は治療薬の効果の評価や判定を行う場合は、大動脈瘤の予防又は治療を必要とする被験対象動物に大動脈瘤の予防薬又は治療薬を投与する前と投与した後、被験対象動物から試料を採取するか、又は当該予防薬又は治療薬を投与した後、被験対象動物から時系列で2以上の時点で試料を採取し、試料におけるMXRA5、TALDO1及びMETRNLのうちいずれか1つ以上のタンパク質レベルの経時変化を調べることにより行うことができる。大動脈瘤の予防薬又は治療薬の投与により、大動脈瘤病変の形成、進展が抑制された場合には、被験対象動物から採取された試料におけるMXRA5、TALDO1及びMETRNLのうちいずれか1つ以上のタンパク質レベルの経時的な増加が抑制又は横ばい傾向を示す。一方、大動脈瘤の予防薬又は治療薬の投与により、大動脈瘤病変が改善された場合には、被験対象動物から採取された試料におけるMXRA5、TALDO1及びMETRNLのうちいずれか1つ以上のタンパク質レベルは経時的な減少傾向を示す。 The marker according to the present invention can also be used for screening for a prophylactic or therapeutic agent for aortic aneurysm, or for evaluating or determining the effect of a prophylactic or therapeutic agent for aortic aneurysm. For example, when evaluating or determining the effectiveness of a prophylactic or therapeutic drug for aortic aneurysm, it may be necessary to After collecting samples from the test animals, or after administering the prophylactic or therapeutic drug, collect samples from the test animals at two or more time points, and determine whether MXRA5, TALDO1, and METRNL in the sample are correct. This can be done by examining changes in the level of one or more proteins over time. If the formation and progression of an aortic aneurysm lesion is suppressed by the administration of a prophylactic or therapeutic drug for aortic aneurysm, one or more proteins among MXRA5, TALDO1, and METRNL in the sample collected from the test animal The increase in levels over time shows a tendency to slow down or level off. On the other hand, if the aortic aneurysm lesion is improved by the administration of a prophylactic or therapeutic agent for aortic aneurysm, the protein level of one or more of MXRA5, TALDO1, and METRNL in the sample collected from the test animal will decrease. It shows a decreasing trend over time.
本発明に係るマーカータンパク質を測定する方法としては、例えば、免疫測定法、質量分析法等、MXRA5、TALDO1及びMETRNLのうちいずれか1つ以上のタンパク質を特異的に測定できる方法であれば周知のいかなる方法を用いても良い。本発明に係るマーカータンパク質を測定する際に用いる検出剤としては、抗体やアプタマー等を用いることができる。 As a method for measuring the marker protein according to the present invention, well-known methods such as immunoassay, mass spectrometry, etc. that can specifically measure any one or more proteins among MXRA5, TALDO1, and METRNL can be used. Any method may be used. As the detection agent used when measuring the marker protein according to the present invention, antibodies, aptamers, etc. can be used.
免疫測定法としては、特に限定されないが、各種のエンザイムイムノアッセイ、ラジオイムノアッセイ(RIA)、酵素結合免疫測定法(ELISA)、二重モノクローナル抗体サンドイッチイムノアッセイ法、モノクローナルポリクローナル抗体サンドイッチアッセイ法、免疫染色法、免疫蛍光法、ウェスタンブロッティング法、ビオチン－アビジン法、免疫沈降法、金コロイド凝集法、イムノクロマト法、ラテックス凝集法(LA)、免疫比濁法(TIA)等を挙げることができる。 Immunoassays include, but are not limited to, various enzyme immunoassays, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), double monoclonal antibody sandwich immunoassays, monoclonal polyclonal antibody sandwich assays, immunostaining methods, Examples include immunofluorescence method, Western blotting method, biotin-avidin method, immunoprecipitation method, colloidal gold agglutination method, immunochromatography method, latex agglutination method (LA), and immunoturbidimetry (TIA).
免疫測定法において使用する検出剤として、既に市販されている抗MXRA5抗体、抗TALDO1抗体、抗METRNL抗体を使用しても良いが、公知のMXRA5のアミノ酸配列(配列番号１)、TALDO1のアミノ酸配列(配列番号２)、METRNLのアミノ酸配列(配列番号３)に基づいて定法により抗体を調製しても良い。MXRA5、TALDO1、METRNLそれぞれのアミノ酸配列の構造を特異的に認識する抗体で良いが、それぞれの糖鎖やジスルフィド結合、リン酸化等の翻訳後修飾を含む全体構造を特異的に認識するものでも良い。 As a detection agent used in the immunoassay, commercially available anti-MXRA5 antibodies, anti-TALDO1 antibodies, and anti-METRNL antibodies may be used, but the known amino acid sequence of MXRA5 (SEQ ID NO: 1) and the amino acid sequence of TALDO1 may be used. (SEQ ID NO: 2) and the amino acid sequence of METRNL (SEQ ID NO: 3), antibodies may be prepared by standard methods. An antibody that specifically recognizes the structure of each amino acid sequence of MXRA5, TALDO1, and METRNL may be used, but it may also be an antibody that specifically recognizes the overall structure of each sugar chain, disulfide bond, post-translational modification such as phosphorylation, etc. .
MXRA5タンパク質、TALDO1タンパク質、METRNLタンパク質を検出可能な抗体であれば由来する動物種やクローンは特に限定されない。ウサギ、ヤギ、マウス、ラット、モルモット、ウマ、ヒツジ、ラクダ、ニワトリ等に由来する抗体が挙げられ、モノクローナル抗体でも、ポリクローナル抗体でも構わない。また、MXRA5タンパク質、TALDO1タンパク質、METRNLタンパク質への特異的な結合に適する全てのサブクラスの抗体を用いることができる。組換え抗体、Fab、Fab'又はF(ab')2断片のような断片を用いることももちろん可能である。 As long as the antibody can detect MXRA5 protein, TALDO1 protein, and METRNL protein, the animal species or clone from which it is derived is not particularly limited. Examples include antibodies derived from rabbits, goats, mice, rats, guinea pigs, horses, sheep, camels, chickens, etc., and monoclonal antibodies or polyclonal antibodies may be used. Furthermore, antibodies of all subclasses suitable for specific binding to MXRA5 protein, TALDO1 protein, and METRNL protein can be used. It is of course also possible to use recombinant antibodies, Fab, Fab' or fragments such as F(ab')2 fragments.
本発明に係る大動脈瘤の検出剤として、MXRA5タンパク質、TALDO1タンパク質、METRNLタンパク質と結合性を有するアプタマーを利用することもできる。アプタマーの製造には、公知のMXRA5のアミノ酸配列(配列番号１)、TALDO1のアミノ酸配列(配列番号２)、METRNLのアミノ酸配列(配列番号３)から、結合し得るDNA若しくはRNA又はそれらの誘導体である核酸アプタマーやアミノ酸で構成されるペプチドアプタマーを周知の方法により合成して用いれば良い。測定の際には、アプタマーの結合を発光法や蛍光法、表面プラズモン共鳴法により検出することができる。 As the aortic aneurysm detection agent according to the present invention, an aptamer that has binding properties to MXRA5 protein, TALDO1 protein, and METRNL protein can also be used. For the production of aptamers, the known amino acid sequence of MXRA5 (SEQ ID NO: 1), the amino acid sequence of TALDO1 (SEQ ID NO: 2), and the amino acid sequence of METRNL (SEQ ID NO: 3) is used to select DNA or RNA that can bind, or a derivative thereof. A certain nucleic acid aptamer or a peptide aptamer composed of amino acids may be synthesized by a well-known method and used. During measurement, aptamer binding can be detected using a luminescence method, a fluorescence method, or a surface plasmon resonance method.
質量分析法としては、特に限定されないが、エレクトロスプレーイオン化法(ESI)、マトリックス支援レーザー脱離イオン化法(MALDI)、表面増強レーザー脱離イオン化法(SELDI)等を用いたイオン源と、飛行時間型分析計(TOF)、イオントラップ型分析計(IT)、フーリエ変換型分析計(FT)等を組み合わせた質量分析計が利用できる。質量分析計を高速液体クロマトグラフィー(HPLC)やキャピラリー電気泳動(CE)等の分離装置と連結したLC-MSやCE-MS等を用いることが出来る。また、質量分析データの取得方法として、データ非依存性解析(DIA)、データ依存性解析(DDA)、多重反応モニタリング法(MRM)等が挙げられる。質量分析では、試料をiTRAQ試薬(SCIEX社)等による安定同位体標識を行う場合も含まれる。 Mass spectrometry methods include, but are not limited to, ion sources using electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), surface-enhanced laser desorption ionization (SELDI), and flight time. Mass spectrometers that combine a type analyzer (TOF), an ion trap type analyzer (IT), a Fourier transform type analyzer (FT), etc. can be used. LC-MS, CE-MS, etc., in which a mass spectrometer is connected to a separation device such as high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE), can be used. Furthermore, methods for acquiring mass spectrometry data include data independent analysis (DIA), data dependent analysis (DDA), multiple reaction monitoring method (MRM), and the like. Mass spectrometry also includes cases in which a sample is labeled with a stable isotope using iTRAQ reagent (SCIEX) or the like.
本発明の大動脈瘤検出を行う際に必要な各種の試薬類は、予めパッケージングしてキット化することができる。例えば、(i)捕捉抗体として本発明に係るマーカータンパク質に特異的なモノクローナル抗体又はポリクローナル抗体、(ii)検出抗体として本発明に係るマーカータンパク質に特異的な酵素標識化モノクローナル抗体又はポリクローナル抗体、(iii)基質溶液等の必要な試薬がキットとして提供される。 Various reagents necessary for detecting an aortic aneurysm according to the present invention can be packaged in advance to form a kit. For example, (i) a monoclonal or polyclonal antibody specific for the marker protein of the present invention as a capture antibody, (ii) an enzyme-labeled monoclonal or polyclonal antibody specific for the marker protein of the present invention as a detection antibody, ( iii) Necessary reagents such as substrate solutions are provided as a kit.
以下、実施例により本発明をより詳細に説明するが、本発明の技術的範囲は以下の実施例に限定されるものではない。 EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to examples, but the technical scope of the present invention is not limited to the following examples.
〔実施例１〕
胸部大動脈瘤患者の血清検体中のMXRA5、TALDO1の測定(MRM法)
MXRA5、TALDO1及び対照としてPRDX1の血清濃度について、多重反応モニタリング法(以下、「MRM法」と称する)による測定を行った。
[Example 1]
Measurement of MXRA5 and TALDO1 in serum samples of patients with thoracic aortic aneurysm (MRM method)
Serum concentrations of MXRA5, TALDO1, and PRDX1 as a control were measured by multiple reaction monitoring method (hereinafter referred to as "MRM method").
詳細には、複数例の血清を混合した後、超遠心機を用いて比重によりLDL/VLDL分画、HDL分画、Protein分画の3分画に分けた。その後、Protein分画については採取後、血中多量タンパク質除去Sepproアフィニティカラム(Sigma-Aldrich社)にて多量タンパク質を除去した。その後、各分画にLys-C(Lysyl Endopeptidase)、Trypsinを添加し、37℃で一晩インキュベーションして消化した。得られたトリプシン消化ペプチドはC18(オクタデシルシリル)カラムを用いて脱塩し、解析サンプルとした。解析サンプルはナノLC(Eksigent EkspertTM nanoLC 425、SCIEX社)で分離した後、連結されたTripleTOF 5600 (SCIEX社)でMS/MS解析を行った。測定データからMascot database search (Matrix science社)でペプチド及びタンパク質情報を取得し、Skylineソフトウェア(University of Washington MacCoss Lab)を用いてMRMメソッド(MRM測定を行うためのプログラム)を作成した。その後、健常者血清及び胸部大動脈瘤患者血清から調製した解析サンプル中のMXRA5、TALDO1、PRDX1の特定のトリプシン消化ペプチドについてMRM法による測定を行い、得られたデータをMultiQuantソフトウェア(SCIEX社)で解析することで、各トリプシン消化ペプチドのピーク面積を比較定量した。 Specifically, sera from multiple cases were mixed and then divided into three fractions, LDL/VLDL fraction, HDL fraction, and Protein fraction, based on specific gravity using an ultracentrifuge. Thereafter, after collecting the protein fraction, large amounts of protein were removed using a Seppro affinity column (Sigma-Aldrich) for removing large amounts of blood proteins. Thereafter, Lys-C (Lysyl Endopeptidase) and Trypsin were added to each fraction, and the mixture was incubated overnight at 37°C for digestion. The obtained trypsin-digested peptides were desalted using a C18 (octadecylsilyl) column and used as analysis samples. The analysis sample was separated using nanoLC (Eksigent Ekspert TM nanoLC 425, SCIEX), and then subjected to MS/MS analysis using a connected TripleTOF 5600 (SCIEX). Peptide and protein information was obtained from the measurement data using Mascot database search (Matrix science), and an MRM method (program for performing MRM measurement) was created using Skyline software (University of Washington MacCoss Lab). Then, specific tryptic peptides of MXRA5, TALDO1, and PRDX1 in analysis samples prepared from serum from healthy subjects and serum from patients with thoracic aortic aneurysm were measured using the MRM method, and the obtained data were analyzed using MultiQuant software (SCIEX). By doing so, the peak areas of each trypsin-digested peptide were comparatively quantified.
・結果1
MXRA5、TALDO1及びPRDX1の測定結果を図１に示す。
解析サンプルは健常人血清23例及び胸部大動脈瘤患者血清8例をそれぞれ混合して調製した。LDL/VLDL分画、HDL分画、Protein分画のそれぞれについて健常人血清に対する胸部大動脈瘤患者血清の当該タンパク質のMRM法による測定を行った特定のトリプシン消化ペプチドのピーク面積比率を示した。MXRA5では、それぞれ90％、94％、446％と、Protein分画において胸部大動脈瘤患者血清でピーク面積が大幅に増加していた。TALDO1では、それぞれ57％、118％、379％と、LDL/VLDL分画では若干低下していたものの、HDL分画、Protein分画において胸部大動脈瘤患者血清でピーク面積が大幅に増加していた。従って、MXRA5、TALDO1が胸部大動脈瘤マーカーとして有用なマーカーであることが示された。
・Result 1
Figure 1 shows the measurement results for MXRA5, TALDO1, and PRDX1.
Samples for analysis were prepared by mixing 23 serum samples from healthy individuals and 8 serum samples from patients with thoracic aortic aneurysm. For each of the LDL/VLDL fraction, HDL fraction, and Protein fraction, the peak area ratios of specific trypsin-digested peptides measured by the MRM method of the relevant proteins in the serum of thoracic aortic aortic aneurysm patients relative to the serum of healthy individuals are shown. In MXRA5, the peak area was significantly increased in serum from patients with thoracic aortic aneurysm in the protein fraction by 90%, 94%, and 446%, respectively. For TALDO1, the peak area was significantly increased in serum from patients with thoracic aortic aneurysm in the HDL fraction and protein fraction, although the LDL/VLDL fraction was slightly decreased to 57%, 118%, and 379%, respectively. . Therefore, MXRA5 and TALDO1 were shown to be useful markers for thoracic aortic aneurysm.
PRDX1についてもそれぞれ、96％、100％、287％と、Protein分画において胸部大動脈瘤患者血清でピーク面積が大幅に増加していた。心血管疾患や大動脈瘤のバイオマーカーとして多数の報告があるPRDX1と、MXRA5、TALDO1が同様に胸部大動脈瘤患者血清でピーク面積の大幅な増加が認められ、MXRA5、TALDO1の胸部大動瘤マーカーとして有用である可能性が強く示された。 Regarding PRDX1, the peak area was significantly increased in the serum of thoracic aortic aortic aneurysm patients in the protein fraction, at 96%, 100%, and 287%, respectively. PRDX1, which has been reported extensively as a biomarker for cardiovascular disease and aortic aneurysm, as well as MXRA5 and TALDO1, similarly showed a significant increase in peak area in the serum of patients with thoracic aortic aneurysm, and MXRA5 and TALDO1 were found to be effective as markers for thoracic aortic aneurysm. It was strongly shown that it could be useful.
・結果2
異なる検体で実施したMXRA5、TALDO1及びPRDX1の測定結果を図２に示す。
解析サンプルは結果1とは異なる健常人血清12例及び胸部大動脈瘤患者血清9例をそれぞれ混合して調製した。LDL/VLDL分画、HDL分画、Protein分画のそれぞれについて健常人血清に対する胸部大動脈瘤患者血清のピーク面積比率を示した。MXRA5では、それぞれ71％、98％、172％と、Protein分画において胸部大動脈瘤患者血清でピーク面積が大幅に増加していた。TALDO1では、それぞれ112％、145％、73％と、Protein分画では若干低下していたものの、LDL/VLDL分画、HDL分画において胸部大動脈瘤患者血清でピーク面積が大幅に増加していた。従って、異なる検体を用いた場合もMXRA5、TALDO1が胸部大動脈瘤マーカーとして有用なマーカーであることが示された。
・Result 2
Figure 2 shows the measurement results of MXRA5, TALDO1, and PRDX1 performed on different samples.
Analysis samples were prepared by mixing 12 serum samples from healthy individuals and 9 serum samples from patients with thoracic aortic aneurysm, which were different from Results 1. The peak area ratios of thoracic aortic aneurysm patient serum to healthy human serum are shown for each of the LDL/VLDL fraction, HDL fraction, and protein fraction. In MXRA5, the peak area was significantly increased in serum from thoracic aortic aortic aneurysm patients in the protein fraction by 71%, 98%, and 172%, respectively. For TALDO1, the peak area was significantly increased in serum from patients with thoracic aortic aneurysm in the LDL/VLDL fraction and HDL fraction, although the protein fraction was slightly decreased to 112%, 145%, and 73%, respectively. . Therefore, it was shown that MXRA5 and TALDO1 are useful markers for thoracic aortic aneurysm even when different samples are used.
PRDX1についてはLDL/VLDL分画では検体量が不足し検出できなかったが、HDL分画、Protein分画における値はそれぞれ98％、99％であった。従って、MXRA5、TALDO1はPRDX1より有用な胸部大動脈瘤マーカーである可能性が示唆された。 PRDX1 could not be detected in the LDL/VLDL fraction due to insufficient sample volume, but the values in the HDL and Protein fractions were 98% and 99%, respectively. Therefore, it was suggested that MXRA5 and TALDO1 may be more useful markers for thoracic aortic aneurysm than PRDX1.
〔実施例２〕
胸部・腹部大動脈瘤患者の血漿検体からのMXRA5の測定(ELISA法)
MXRA5の血漿濃度について、ELISA測定を行った。MXRA5は、ELISA Kit for Matrix Remodeling Associated Protein 5 (cloud-clone社)を用いて測定した。測定はキット付属のプロトコールに従って実施した。
[Example 2]
Measurement of MXRA5 from plasma samples of patients with thoracic/abdominal aortic aneurysm (ELISA method)
ELISA measurements were performed on the plasma concentration of MXRA5. MXRA5 was measured using ELISA Kit for Matrix Remodeling Associated Protein 5 (cloud-clone). Measurements were performed according to the protocol included with the kit.
詳細には、抗MXRA5抗体の固相化されたプレートに、希釈した血漿検体を添加してインキュベーションした。その後、検体液を取り除き、検出試薬A液を添加してさらにインキュベーションした。インキュベーション後に洗浄操作を行い、検出試薬B液を添加してインキュベーションした。続いて洗浄操作を行い、TMB(3,3',5,5'-tetramethylbenzidine)溶液を添加して発色反応を行った後、停止液を添加し、マイクロプレートリーダー(SpectraMax M2e、Molecular Devices社)を用いて主波長450nm、副波長540nmで測定を行った。そのデータを、同時に測定した各標準液の測定データから作成した検量線から濃度を算出し、MXRA5濃度を測定した。統計処理には、StatFlex ver6.0(アーテック社)を使用した。 Specifically, a diluted plasma sample was added to a plate immobilized with anti-MXRA5 antibody and incubated. Thereafter, the sample solution was removed, detection reagent A solution was added, and further incubation was performed. After incubation, a washing operation was performed, and detection reagent B solution was added, followed by incubation. Next, a washing operation is performed, a TMB (3,3',5,5'-tetramethylbenzidine) solution is added to perform a color reaction, a stop solution is added, and a microplate reader (SpectraMax M2e, Molecular Devices) is used. Measurements were performed using a main wavelength of 450 nm and a sub wavelength of 540 nm. The concentration was calculated from a calibration curve created from the measurement data of each standard solution measured at the same time, and the MXRA5 concentration was measured. StatFlex ver6.0 (Artec) was used for statistical processing.
・結果1
MXRA5の結果を図３に示す。
図３に示す結果から、MXRA5は健常者(15例)及び大動脈瘤患者(胸部、腹部含む34例)でそれぞれ、23.9±12.3ng/mL、28.8±12.6ng/mLと、大動脈瘤患者で血漿MXRA5値が増加していた。従って、MRM法による測定結果(実施例１)と一致して、MXRA5が大動脈瘤マーカーとして有用なマーカーであることが示された。
・Result 1
The results of MXRA5 are shown in Figure 3.
From the results shown in Figure 3, MXRA5 was 23.9 ± 12.3 ng/mL and 28.8 ± 12.6 ng/mL in healthy subjects (15 cases) and aortic aneurysm patients (34 cases including chest and abdomen), and plasma in aortic aneurysm patients. MXRA5 value was increasing. Therefore, consistent with the measurement results by the MRM method (Example 1), MXRA5 was shown to be a useful marker as an aortic aneurysm marker.
・結果2(ROC曲線によるMXRA5の大動脈瘤マーカーとしての評価)
大動脈瘤の診断におけるMXRA5のROC(Receiver Operating Characteristic)曲線を図４に示した。ROC曲線は統計解析ソフトStatFlex ver6.0(アーテック社)で作成した。ROC曲線から求めたAUC(Area Under the Curve)は、0.645であった。従って、MXRA5は大動脈瘤マーカーとして有用なマーカーであることが示された。
・Result 2 (Evaluation of MXRA5 as an aortic aneurysm marker using ROC curve)
Figure 4 shows the ROC (Receiver Operating Characteristic) curve of MXRA5 in diagnosis of aortic aneurysm. The ROC curve was created using the statistical analysis software StatFlex ver. 6.0 (Artec Inc.). AUC (Area Under the Curve) determined from the ROC curve was 0.645. Therefore, MXRA5 was shown to be a useful aortic aneurysm marker.
〔実施例３〕
胸部大動脈瘤患者の血清検体からのMETRNLの測定(ELISA法)と、ROC曲線による胸部大動脈瘤マーカーとしての評価
METRNLの血清濃度について、ELISA測定を行った。METRNLは、Human Meteorin-like/METRNL DuoSet ELISA (R&D Systems社)を用いて測定した。測定はキット付属のプロトコールに従って実施した。
[Example 3]
Measurement of METRNL from serum samples from thoracic aortic aneurysm patients (ELISA method) and evaluation as a thoracic aortic aneurysm marker using ROC curves
The serum concentration of METRNL was measured by ELISA. METRNL was measured using Human Meteorin-like/METRNL DuoSet ELISA (R&D Systems). Measurements were performed according to the protocol included with the kit.
詳細には、抗METRNL抗体の固相化されたプレートに、希釈した血清検体を添加してインキュベーションした。その後、検体液を取り除き、ペルオキシダーゼが結合した抗METRNL抗体を添加してインキュベーションした。インキュベーション後に洗浄操作を行い、発色反応を行った後、停止液を添加し、マイクロプレートリーダー(SpectraMax M2e、Molecular Devices社)を用いて主波長450nm、副波長540nmで測定を行った。そのデータを、同時に測定した各標準液の測定データから作成した検量線から濃度を算出し、METRNL濃度を測定した。統計処理には、StatFlex ver6.0(アーテック社)を使用し、有意差検定にはMann-Whitney U検定を用いた。 Specifically, a diluted serum sample was added to a plate immobilized with anti-METRNL antibody and incubated. Thereafter, the sample solution was removed, and peroxidase-conjugated anti-METRNL antibody was added and incubated. After incubation, a washing operation was performed, a color reaction was performed, a stop solution was added, and measurement was performed using a microplate reader (SpectraMax M2e, Molecular Devices) at a main wavelength of 450 nm and a sub wavelength of 540 nm. The concentration was calculated from a calibration curve created from the measurement data of each standard solution measured at the same time, and the METRNL concentration was measured. StatFlex ver6.0 (Artec) was used for statistical processing, and the Mann-Whitney U test was used for significance testing.
・結果1
METRNLの結果を図５に示す。
図５に示す結果から、METRNLは健常者(41例)及び胸部大動脈瘤患者(28例)でそれぞれ、1318.2±189.4 pg/mL、1629.6±475.8 pg/mLと、有意に胸部大動脈瘤患者で血清METRNL値が増加していた。従って、METRNLが胸部大動脈瘤マーカーとして有用なマーカーであることが示された。
・Result 1
The results of METRNL are shown in Figure 5.
From the results shown in Figure 5, METRNL was 1318.2 ± 189.4 pg/mL and 1629.6 ± 475.8 pg/mL in healthy subjects (41 cases) and thoracic aortic aneurysm patients (28 cases), respectively, which was significantly higher in serum patients with thoracic aortic aneurysm patients. METRNL values were increasing. Therefore, METRNL was shown to be a useful marker for thoracic aortic aneurysm.
・結果2(ROC曲線によるMETRNLの胸部大動脈瘤マーカーとしての評価)
胸部大動脈瘤の診断におけるMETRNLのROC(Receiver Operating Characteristic)曲線を図６に示した。ROC曲線は統計解析ソフトStatFlex ver6.0(アーテック社)で作成した。ROC曲線から求めたAUC(Area Under the Curve)は、0.731であった。従って、METRNLは胸部大動脈瘤マーカーとして有用なマーカーであることが示された。
・Result 2 (Evaluation of METRNL as a thoracic aortic aneurysm marker using ROC curve)
Figure 6 shows the ROC (Receiver Operating Characteristic) curve of METRNL in the diagnosis of thoracic aortic aneurysm. The ROC curve was created using the statistical analysis software StatFlex ver. 6.0 (Artec Inc.). AUC (Area Under the Curve) determined from the ROC curve was 0.731. Therefore, METRNL was shown to be a useful marker for thoracic aortic aneurysm.
〔実施例４〕
腹部大動脈瘤患者の血清検体からのMETRNLの測定(ELISA法)と、ROC曲線による腹部大動脈瘤マーカーとしての評価
METRNLの血清濃度について、ELISA測定を行った。METRNLは、Human Meteorin-like/METRNL DuoSet ELISA (R&D Systems社)を用いて測定した。測定はキット付属のプロトコールに従って実施した。
[Example 4]
Measurement of METRNL from serum samples from abdominal aortic aneurysm patients (ELISA method) and evaluation as an abdominal aortic aneurysm marker using ROC curves
The serum concentration of METRNL was measured by ELISA. METRNL was measured using Human Meteorin-like/METRNL DuoSet ELISA (R&D Systems). Measurements were performed according to the protocol included with the kit.
詳細には、抗METRNL抗体の固相化されたプレートに、希釈した血清検体を添加してインキュベーションした。その後、検体液を取り除き、ペルオキシダーゼが結合した抗METRNL抗体を添加してインキュベーションした。インキュベーション後に洗浄操作を行い、発色反応を行った後、停止液を添加し、マイクロプレートリーダー(SpectraMax M2e、Molecular Devices社)を用いて主波長450nm、副波長540nmで測定を行った。そのデータを、同時に測定した各標準液の測定データから作成した検量線から濃度を算出し、METRNL濃度を測定した。統計処理には、StatFlex ver6.0(アーテック社)を使用し、有意差検定にはMann-Whitney U検定を用いた。 Specifically, a diluted serum sample was added to a plate immobilized with anti-METRNL antibody and incubated. Thereafter, the sample solution was removed, and peroxidase-conjugated anti-METRNL antibody was added and incubated. After incubation, a washing operation was performed, a color reaction was performed, a stop solution was added, and measurement was performed using a microplate reader (SpectraMax M2e, Molecular Devices) at a main wavelength of 450 nm and a sub wavelength of 540 nm. The concentration was calculated from a calibration curve created from the measurement data of each standard solution measured at the same time, and the METRNL concentration was measured. StatFlex ver6.0 (Artec) was used for statistical processing, and the Mann-Whitney U test was used for significance testing.
・結果1
METRNLの結果を図７に示す。
図７に示す結果から、METRNLは健常者(41例)及び腹部大動脈瘤患者(38例)でそれぞれ、1318.2±189.4 pg/mL、1542.4±406.0 pg/mLと、有意に腹部大動脈瘤患者で血清METRNL値が増加していた。従って、METRNLが腹部大動脈瘤マーカーとして有用なマーカーであることが示された。
・Result 1
The results of METRNL are shown in Figure 7.
From the results shown in Figure 7, METRNL was 1318.2 ± 189.4 pg/mL and 1542.4 ± 406.0 pg/mL in healthy subjects (41 cases) and abdominal aortic aneurysm patients (38 cases), respectively, which was significantly higher in patients with abdominal aortic aneurysm. METRNL values were increasing. Therefore, METRNL was shown to be a useful marker for abdominal aortic aneurysm.
・結果2(ROC曲線によるMETRNLの腹部大動脈瘤マーカーとしての評価)
腹部大動脈瘤の診断におけるMETRNLのROC(Receiver Operating Characteristic)曲線を図８に示した。ROC曲線は統計解析ソフトStatFlex ver6.0(アーテック社)で作成した。ROC曲線から求めたAUC(Area Under the Curve)は、0.653であった。従って、METRNLは腹部大動脈瘤マーカーとして有用なマーカーであることが示された。
・Result 2 (Evaluation of METRNL as an abdominal aortic aneurysm marker using ROC curve)
Figure 8 shows the ROC (Receiver Operating Characteristic) curve of METRNL in the diagnosis of abdominal aortic aneurysm. The ROC curve was created using the statistical analysis software StatFlex ver. 6.0 (Artec Inc.). AUC (Area Under the Curve) determined from the ROC curve was 0.653. Therefore, METRNL was shown to be a useful marker for abdominal aortic aneurysm.
本発明に係る大動脈瘤の新規マーカーであるMXRA5、TALDO1及びMETRNLのうちいずれか1つ以上のタンパク質を用いることにより、大動脈瘤の発症を高感度で特異的に検出することができる。そのため、本発明に係る大動脈瘤の検出剤及び検出キットは、大動脈瘤のスクリーニングや早期診断の補助、予防薬や治療薬の開発、評価等に使用することができ、また、本発明は、当該検出剤及び検出キットを製造する産業において利用することができる。 By using one or more of the novel markers of aortic aneurysm, MXRA5, TALDO1, and METRNL according to the present invention, the onset of aortic aneurysm can be detected specifically and with high sensitivity. Therefore, the aortic aneurysm detection agent and detection kit according to the present invention can be used to assist in screening and early diagnosis of aortic aneurysms, and to develop and evaluate preventive and therapeutic drugs. It can be used in industries that manufacture detection agents and detection kits.
Claims (14)
1) MXRA5(Matrix-remodeling-associated protein 5)タンパク質、
2) TALDO1(Transaldolase)タンパク質、
3) METRNL(Meteorin-like protein)タンパク質。 an aortic aneurysm , the sample being selected from the group consisting of serum, plasma, and blood, comprising the step of measuring any one or more of the following proteins in 1) to 3) in a sample obtained from the subject; A method for detecting .
1) MXRA5 (Matrix-remodeling-associated protein 5) protein,
2) TALDO1 (Transaldolase) protein,
3) METRNL (Meteorin-like protein) protein.
1) MXRA5(Matrix-remodeling-associated protein 5)タンパク質、
2) TALDO1(Transaldolase)タンパク質、
3) METRNL(Meteorin-like protein)タンパク質。 an aortic aneurysm , the sample being selected from the group consisting of serum, plasma, and blood, comprising the step of measuring any one or more of the following proteins in 1) to 3) in a sample obtained from the subject; A method for detecting or screening subjects at risk of developing a disease in the future.
1) MXRA5 (Matrix-remodeling-associated protein 5) protein,
2) TALDO1 (Transaldolase) protein,
3) METRNL (Meteorin-like protein) protein.
1) MXRA5(Matrix-remodeling-associated protein 5)タンパク質、
2) TALDO1(Transaldolase)タンパク質、
3) METRNL(Meteorin-like protein)タンパク質。 An aortic aneurysm detection marker containing one or more of the following proteins 1) to 3).
1) MXRA5 (Matrix-remodeling-associated protein 5) protein,
2) TALDO1 (Transaldolase) protein,
3) METRNL (Meteorin-like protein) protein.
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| US20110281374A1 (en) | 2010-05-12 | 2011-11-17 | Rainger George Edward | Biomarker |
| JP2016217965A (en) | 2015-05-22 | 2016-12-22 | 公立大学法人横浜市立大学 | Method for determining disease activity of non-dissecting aortic aneurysm |
| JP2019032334A (en) | 2018-10-03 | 2019-02-28 | イムノヴィア・アクチエボラーグ | Method for determining breast cancer-related disease state, and array to be used in the method |
| WO2019189744A1 (en) | 2018-03-30 | 2019-10-03 | 栄研化学株式会社 | Novel aortic aneurysm marker |
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| US20110281374A1 (en) | 2010-05-12 | 2011-11-17 | Rainger George Edward | Biomarker |
| JP2016217965A (en) | 2015-05-22 | 2016-12-22 | 公立大学法人横浜市立大学 | Method for determining disease activity of non-dissecting aortic aneurysm |
| WO2019189744A1 (en) | 2018-03-30 | 2019-10-03 | 栄研化学株式会社 | Novel aortic aneurysm marker |
| JP2019032334A (en) | 2018-10-03 | 2019-02-28 | イムノヴィア・アクチエボラーグ | Method for determining breast cancer-related disease state, and array to be used in the method |
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